1. Field of the Invention
This invention relates to methods for rationally designing cell culture media for use in cell cultures employed in, e.g., polypeptide production; cell culture media designed with the disclosed methods; methods of producing large quantities of a polypeptide of interest, e.g., an antibody, using such media; polypeptides produced using the methods and media disclosed herein; and pharmaceuticals compositions containing such polypeptides. The invention is particularly useful in large-scale cell cultures. The methods and compositions disclosed herein are particularly useful to produce significant quantities of polypeptides in batch, fed-batch and perfusion animal cell cultures.
2. Related Background Art
A large proportion of biotechnology products, whether commercially available or only in development, are protein therapeutics; thus, there is a demand for production of these polypeptides in cell cultures. Furthermore, the cellular machinery of an animal cell (as opposed to, e.g., a bacterial cell) is often required to produce many forms of polypeptide therapeutics (such as glycosylated proteins or hybridoma-produced monoclonal antibodies (MAbs)). Consequently, there is an increasing demand for optimizing production of these polypeptides in cell cultures, and particularly in animal cell cultures.
As compared to bacterial cell cultures, animal cell cultures have lower production rates and typically generate lower production yields. Thus, a significant quantity of research focuses on animal cell culture conditions that optimize the polypeptide output, i.e., conditions that support high cell density and high titer. For example, it has been determined that maintaining glucose concentrations in cell culture media at low concentrations and culturing cells in a production phase at an osmolality of about 400 to 600 mOsm increases production of recombinant proteins by animal cell cultures, wherein culturing in all phases is also at a selected glutamine concentration (preferably between about 0.2 to about 2 mM). It has also been determined that restricted feeding of glucose to animal cell cultures in fed-batch processes controls lactate production without requiring the constant-rate feeding of glucose. Further, it is known that modification of the total cumulative concentration of amino acids, the concentration of individual amino acids, and the ratios of individual amino acids to each other (e.g., glutamine to asparagine) and to total amino acids (e.g., glutamine to total amino acids) in the media of a large-scale cell culture can result in substantially improved large-scale polypeptide production.
Traditionally, medium studies for animal cell cultures focus on three techniques: 1) enriching the medium components of the starting medium and increasing the frequency of culture feeding; 2) applying multi-factorial design to different medium strengths and different component concentrations; and 3) analyzing conditioned (spent) medium for amino acids, vitamins, and other components, and adding those components that are at low levels or are depleted. These methods generally use cell density, viability and titer responses as indicators of optimization.
However, the above methods only indirectly detect the nutrient requirement for cells based on the end result, i.e., cell density, viability, and titer, rather than detecting and providing the cell with the actual nutrient requirement for optimized protein production.